This periodical project scientific report covers the work progress delivered by NIBIO team (Norwegian partner of BioShell project) since 01.10.2020. Under the continuation of global pandemic in year 2021, numbers of planned tasks such as wastewater sampling campaigns and lab experiments have been affected and left behind the schedule with certain delay. However, by side of our Romania partner (project coordinator) striving on methodological development of chitosan preparation from the raw materials, NIBIO team determinedly conducted a number of research and experimental activities. Our main focus was to screen the microbial hazards, including prevalent waterborne pathogens (WBPs) and antibiotic resistance genes (ARGs) in wastewater (WW) samples delivered from Romania. For this, we used our established molecular diagnostic toolkit containing the verified genetic markers for all target WBPs and ARGs. Moreover, the developed ARG markers served to evaluate the ARG mitigation efficacy of the newly generated functional nanomaterials and the promising results have been published in an international peer-reviewed journal Polymers (IF 4.3).
The WW samples were collected weekly at the inlet of a local wastewater treatment plant (WWTP). The purpose of their examination was to obtain a background information on the extend and degree of the WBPs and ARGs contents in the raw WW, and thus better inform the future investigation (evaluation of the WW purification efficiency of the developed nanoproducts). WBP screening results indicated that except Campylobacter jejuni (C. jejuni) all other pathogens were detected in the WW samples. Among them, in terms of gene copy numbers in 100mL WW, Salmonella typhimurium (S. typhimurium) were discovered in highest abundance (4.5 x 106 ? 3.4 x 109), thereafter followed by Shiga toxin producing E. coli (STEC) (stx1: 6.6 x 105 - 1.3 x 106; stx2: 4.8 ? 5.1 x 104; eae: 6.2 x 102 ? 1.7 x 103), Enterococcus faecalis (E. faecalis) at 9.8 x 103 ? 3.8 x 104, Clostridium perfringens (C. perfringens) at 6.9 x 102 ? 1.4 x 104, Legionella pneumophila (L. pneumophila) at 2.7 x 102 ? 1.7 x 103, and Cryptosporidium parvum (C. parvum) at 2.4 x 102 ? 2.0 x 103. When it comes to the ARGs profiling, qnrS (encoding resistance to Fluoroquinolones) was the most prevalent ARG in the studied WW with concentration at 2.3 x 108 ? 2.4 x 109 / 100mL. In the following rank, sul1 (resistance to Sulfonamide), tetO (resistance to tetracycline) and ermB (resistance to Erythromycin/Macrolides) were also remarkably abundant in WW, at 6.1 x 105 ? 3.1 x 106, 1.5 ? 6.1 x 106 and 1.0 ? 5.7 x 105, respectively. Notably, intl1 (Mobile Genetic Element/conferring Multidrug Resistance), was found also in high content (2.4 ? 4.6 x 106), implying a considerable potential for broad ARG spread via horizontal gene transfer. Additionally, mecA (resistance to Methicilin) and blaCTXM (resistance to Cefotaxime, Ceftazidime/?-lactam) were detected at 2.4 x 102 ? 1.7 x 103 and 4.4 x 102 ? 1.7 x 104, respectively. Yet, vanA conferring resistance to Vancomycin was not detected in either analyzed WW sample, indicating its absence/at very low amount (below the detection limit of the assay) in WW. This corresponds with our previous findings and is likely due to Vancomycin being the last-resort antibiotics with limited prescription/usage and discharge into WW.
The developed ARG markers were further used for investigation on the efficiency of ARGs removal from WW being treated by the first generation of functionalized nanocomposites created by our Romania partner. These novel nanomaterials constitute molecular imprinted polymer (MIP) films and quaternary ammonium salt (QAS) modified kaolin microparticles. They have demonstrated significant bactericidal activity against WBPs in WW and therefore, we sought out to explore their ARGs mitigation potential using the newly developed ARG markers. The results were striking and promising, target ARGs prevalent usually in WWTPs have been removed/eliminated from WW after sequential treatment by MIP and QAS-kaolin. The outcomes revealed that blaCTXM, ermB and qnrS, can be drastically reduced by 2.7, 3.9 and 4.9 log (copies/100 mL), respectively, whereas sul1, tetO and mecA were eliminated below their detection limits. Intl1 was reduced by 4.3 log. Taken altogether, we demonstrated that the application of these new nanomaterials has great potential in mitigating the major health critical risks from WBPs and ARGs present in WW. The achieved results have been published as: Paruch L., Paruch A.M., Iordache T-V, Olaru A.G., Sarbu A. 2021. Mitigating antibiotic resistance genes in wastewater by sequential treatment with novel nanomaterials. Polymers, 13(10), 1593. https://doi.org/10.3390/polym13101593.
Wastes from agriculture and fishery cause harmful effects on the environment and implicitly on humans. Yet, many of these wastes can be recycled. One of the current global issues refers to minimizing waste production, effective wastewater treatment, biosafe food production, and reducing hazards from the exposure to pathogens. Most of the threatening microorganisms, especially Emerging Pathogens (EPs), derive from wastewater. Moreover, antibiotics residues present in wastewater lead bacterial pathogens to develop Antibiotic Resistance Genes (ARGs). In addition, heavy metals are among the most harmful non-microbial pollutants due to their toxicity to humans.
The BIOSHELL project aims at synergistically solving economic, environmental and health problems caused by agricultural and food industry wastes. The project focuses on utilizing the wastes from seafood preparation, such as crustacean carcasses, in the development of innovative and efficient inorganic-organic functionalized hydrogel nanocomposites, suitable to facilitate the sustainable wastewater purification technologies for heavy metals retention, antibiotics elimination, EPs and ARGs removal.
Functional biopolymer-based hydrogels starting from valorized crustacean's shell wastes will be developed both for the metal and antibiotics retention in waters as well as for anti-bacterial treatment. These competitive materials will be Ion Imprinted Polymers (IIPs) or Molecularly Imprinted Polymers (MIPs). They will benefit from new synthesis methodologies applied for chelating the chitosan nanocomposites and for the chemical grafting of the bactericidal hybrid surfaces. The development of new approaches for the valorization of crustacean wastes, by the new functionalized bio-hydrogels, will improve water purification and wastewater treatment in general, but particularly for decentralised locations/on-site systems. The regeneration of new bio-based agents shall also be targeted.